Enhancers are transcriptional regulatory sequences that modulate gene expression from distant genomic sites. Such sequences are typically marked by DNase1 hypersensitive sites (HS), H3K27ac histone modifications, and co-activators such as p300. Clusters of DNase1 HS having these characteristics are referred to as super-enhancers and implicated in human disease. Remarkably, 80% of human disease-associated single nucleotide polymorphisms (SNPs) are located in or close to enhancer-like genomic sequences. While enhancers are known to bind multiple transcription factors, it is unclear how transcription factor binding confers the many different properties of enhancers. We refer to this combinatorial functionality as the enhancer code. The goals of this project are to gain insight into the enhancer code using IgH and TCR enhancers as paradigms. During FY19 we found that: 1)Point mutations in individual motifs (transcription factor binding sites) of E did not significantly affect E function as measured by B cell development, germline transcription and VDJ recombination in primary bone marrow pro-B cells and CD4+CD8+ thymocytes. Maximum effect was seen with a double mutation of mE2 and mE5 sites that bind E47. 2)Conversely, IgH alleles in which the 700bp E was substituted by multimerized TetO and Gal4 binding sites behaved like an E null allele as measured by the 3 assays in (1) above. Ongoing studies aim to recruit WT E2A and deletion mutations of this protein to determine how much enhancer activity can be substituted by the protein alone. 3)We generated E2E5mAmB-quadruple-mutated mice in which binding sites for both E47 and ETS domain proteins were eliminated from E. Functional assays are underway to explore the functional consequences of this mutation.